Method for detecting target nucleic acid

ABSTRACT

A target nucleic acid having a target sequence in a sample is detected according to the steps of: (a) mixing a first probe including a nucleic acid which has a specific region having a sequence complementary to the target sequence and a nonspecific region having a sequence that is not complementary to the target sequence of the target nucleic acid; a second probe including a nucleic acid which has a first region that is complementary to at least a portion of the nonspecific region of the first probe, a loop region that does not have a sequence complementary to the first probe, and a second region that is complementary to at least a portion of the specific region of the first probe, the loop region being capable of forming a loop when it is annealed with the first probe, wherein the nucleic acid is labeled with a labeling material generating a signal by which formation of the aforementioned loop can be detected; and a sample under conditions in which the first probe and the second probe are annealed and the first probe and the target nucleic acid are annealed; and (b) detecting a signal of the labeling material.

RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/JP03/05773, filed May 8, 2003, which waspublished in a language other than English and which claims the priorityof JP 2002-132995, filed May 8, 2002.

TECHNICAL FIELD

The present invention relates to a method and kit for detecting a targetnucleic acid having a target sequence in a sample. The method and kit ofthe present invention permit the target nucleic acid to be detected inreal time and are useful in fields of biochemistry and so forth.

BACKGROUND ART

A method for detecting a target nucleic acid having a target sequence ina sample, which has been used, includes a hybridization method in whicha probe is used, a PCR method in which oligonucleotide primers are used,and other methods. Further, the PCR method is generally used in variousfields including the detection and cloning of target nucleic acid, andvarious improved methods have been developed.

A so-called real time PCR has been known, which is a PCR method thatperforms amplification of a target sequence and analysis of theamplified product simultaneously. Means for analyzing the amplifiedproducts that has been known include, for example, a Taq-Man probemethod (U.S. Pat. No. 5,210,015 A, JP 06-500021 A, and Holland et al.,Pro. Natl. Aca. Sci. USA., 88, 7276-7280, 1991), a molecular beaconmethod (JP 05-123195 A, Sanjay Tyagi et al., Nature Biotechnology, vol14, March 1996), an intercalator method (Bio/Technology, 10, 413-417,1992, Bio/Technology, 11, 1026-1030, 1993, and JP05-237000 A), and thelike.

In the Taq-Man probe method, a fluorescent material and a probe labeledwith a quencher that quenches fluorescence emitted by the fluorescentmaterial are used. When the probe is hybridized with a target nucleicacid, the quencher quenches the fluorescence while the probe is cleavedby the 5′→3′ exonuclease activity of the polymerase used in PCR at thetime of amplification reaction. As a result, the fluorescent material isreleased from the quencher to emit fluorescence. The amount of thedouble stranded DNA molecule can be known from this fluorescence.

Further, the molecular beacon method is a method that uses a probeincluding a sequence complementary to a target sequence and an armhaving sequences complementary to each other at both sides thereof aswell as a fluorescent material and a quencher bonded to both the ends.When the probe is annealed to the target nucleic acid, the fluorescentmaterial emits fluorescence while when the probe is dissociated from thetarget nucleic acid, the probe forms an arm resulting in that thefluorescent material and the quencher become closer to each other tocause quenching.

On the other hand, the intercalator method is a method that detects adouble stranded DNA using an intercalator such as ethidium bromide.

Although the methods for quantifying PCR amplified products in real timehave been known as described above, they have problems; the Taq-Manprobe method cannot be applied in the case of amplification methods thatuse polymerases having no 5′→3′ exonuclease activity, the molecularbeacon method is difficult to design a probe and suffers poor detectionefficiency due to the intermolecular bond, and the intercalator methodhas no sequence specificity.

DISCLOSURE OF THE INVENTION

The present invention has been made from the aforementioned viewpoint,and it is an object of the present invention to provide a method and kitfor quantifying a target nucleic acid in real time and in a simplemanner without using polymerases having 5′→3′ exonuclease activity.

The inventors of the present invention have made extensive studies inorder to achieve the aforementioned object and as a result they havefound that use of two types of probes differing in length enablesquantification of a target nucleic acid in a simple manner, therebyaccomplishing the present invention.

That is, the present invention relates to:

(1) A method for detecting a target nucleic acid having a targetsequence in a sample, comprising the steps of:

(a) mixing a first probe including a nucleic acid which has a specificregion having a sequence complementary to the target sequence and anonspecific region having a sequence that is not complementary to thetarget sequence of the target nucleic acid; a second probe including anucleic acid which has a first region that is complementary to at leasta portion of the nonspecific region of the first probe, a loop regionthat does not have a sequence complementary to the first probe, and asecond region that is complementary to at least a portion of thespecific region of the first probe, the loop region being capable offorming a loop when it is annealed with the first probe, wherein thenucleic acid is labeled with a labeling material generating a signal bywhich formation of the aforementioned loop can be detected; and a sampleunder conditions in which the first probe and the second probe areannealed and the first probe and the target nucleic acid are annealed;and

(b) detecting a signal of the labeling material.

(2) A method according to item (1), wherein the second region of thesecond probe is shorter than the specific region of the first probe.

(3) A method according to item (1) or (2), wherein the labeling materialcomprises a fluorescent material and a quencher that quenches thefluorescence of the fluorescent material when the quencher is near thefluorescent material, arranged so as to sandwich the loop region, withthe fluorescence of the fluorescent material being quenched by thequencher when the first probe and the second probe are annealed to formthe loop and not quenched when the first probe and the second probe arenot annealed as compared when the probes are annealed.

(4) A method according to any one of items (1) to (3), wherein thedetection of the signal is performed quantitatively, thereby quantifyingthe target nucleic acid.

(5) A kit for detecting a target nucleic acid having a target sequencein a sample, comprising a first probe including a nucleic acid which hasa specific region having a sequence complementary to the target sequenceand a nonspecific region having a sequence that is not complementary tothe target sequence of the target nucleic acid; a second probe includinga nucleic acid which has a first region that is complementary to atleast a portion of the nonspecific region of the first probe, a loopregion that does not have a sequence complementary to the first probe,and a second region that is complementary to at least a portion of thespecific region of the first probe, the loop region being capable offorming a loop when it is annealed with the first probe, wherein thenucleic acid is labeled with a labeling material generating a signal bywhich formation of the aforementioned loop can be detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is schematic diagrams illustrating the concept of the presentinvention, with F indicating a fluorescent material and Q indicating aquencher.

FIG. 2 is a graph showing the time-course changes of the intensities offluorescence of the reaction mixtures (not subjected to heat treatment),where

-   -   solid line: Probe 1+Probe 2+target oligonucleotide    -   dotted line: Probe 1+Probe 2    -   alternate longer and shorter dashed lines: Probe 2.

FIG. 3 is a graph showing the time-course changes of intensities offluorescence of the reaction mixtures (subjected to heat treatment).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The method of the present invention is a method for detecting a targetnucleic acid having a target sequence in a sample. The target nucleicacid is not particularly limited so far as it has the target sequence;it may be either a DNA or an RNA, and it may be either single strandedor double stranded. The present invention is advantageously applied todetection of particularly a double stranded DNA. In a preferable mode ofthe present invention, the target nucleic acid is detectedquantitatively. Note that quantitative detection includes measurement ofan absolute amount of a nucleic acid and measurement of a nucleic acidrelatively to a certain amount.

The target sequence is usually a sequence specific to the target nucleicacid and is not particularly limited in sequence and length so far as itcan form a specific hybrid with a probe having a sequence complementaryto that sequence. The length of the target sequence is preferably 6bases or more, more preferably 15 bases or more.

Samples that contain the target nucleic acid are not particularlylimited and include nucleic acids or nucleic acid mixtures extractedfrom cells or tissues, and PCR nucleic acid amplification reactionmixtures using such nucleic acids or nucleic acid mixtures as templates.

In the present invention, two types of probes are used in detecting thetarget nucleic acid (see FIG. 1). A first probe (hereinafter, alsoreferred to as “Probe 1”) includes a nucleic acid which has a specificregion having a sequence complementary to the target sequence and anonspecific region having a sequence that is not complementary to thetarget sequence of the target nucleic acid (FIG. 1A). Due to such astructure, when Probe 1 is hybridized with the target nucleic acid, thenonspecific region remains as a single strand and forms a flap (FIG.1B). The length of the nonspecific region is preferably 10 bases ormore, more preferably 10 to 30 bases.

A second probe (hereinafter, also referred to as “Probe 2”) includes anucleic acid which has a first region that is complementary to at leasta portion of the aforementioned nonspecific region of the Probe 1, aloop region that does not have a sequence complementary to Probe 1, anda second region that is complementary to at least a portion of thespecific region of Probe 1, the loop region being capable of forming aloop when it is annealed with Probe 1, and the nucleic acid is labeledwith a labeling material generating a signal by which formation of theaforementioned loop can be detected (FIG. 1A).

Note that FIG. 1 shows examples of Probe 1 that has the specific regionon the 5′ side and the nonspecific region on the 3′ side and of Probe 2that has the first region, the loop region, and the second region inorder from the 5′ side. In the present invention, Probe 1 may have thespecific region on the 3′ side and the nonspecific region on the 5′ sideand Probe 2 may have the first region, the loop region, and the secondregion in order from the 3′ side.

The first region has a sequence that is complementary to at least aportion of the nonspecific region of Probe 1 and preferably has asequence that is complementary to the whole nonspecific region. On theother hand, the second region has a sequence that is complementary to atleast a portion of the specific region of Probe 1 and preferably isshorter than the specific region. By making the second region shorterthan the specific region, Probe 1 can be annealed with the targetnucleic acid preferentially than Probe 2. The length of the secondregion is preferably 6 bases or more, more preferably 6 to 20 bases.Alternatively, it is desirable that the second region is made shorter bypreferably one base or more, more preferably 4 bases or more than thespecific region.

The loop region is a region that does not have a sequence complementaryto Probe 1 and preferably it does not have a sequence complementary tothe target nucleic acid. The loop region forms a protruded portion inthe form of a loop when Probe 2 is bonded to Probe 1 but when Probe 2and Probe 1 are dissociated from each other, the loop structure iseliminated (FIG. 1C). Probe 2 is labeled with a labeling materialgenerating a signal by which formation of a loop can be detected. Thelabeling material specifically includes, for example, an energy donorand an energy receptor arranged so as to sandwich the loop region. Theenergy donor and energy receptor include, for example, a fluorescentmaterial and a quencher that quenches the fluorescence generated by thefluorescent material. The quencher quenches fluorescence when it is nearthe fluorescent material but it will no longer quench fluorescence whenthe distance between the fluorescent material and the quencher is equalto or greater than a certain distance. With such a fluorescent materialand quencher, the fluorescence of the fluorescent material is quenchedby the quencher when Probe 1 and Probe 2 are annealed to form a loopwhile the fluorescence is not quenched when Probe 1 and Probe 2 are notannealed. Therefore, by measuring the fluorescence from the fluorescentmaterial, formation of a loop, that is the state of hybridization ofProbe 1 and Probe 2 can be detected. Examples of the fluorescentmaterial include fluorescein dyes such as fluorescein and fluoresceinisothiocyanate (FITC) and examples of the quencher include rhodaminedyes such as tetramethyl rhodamine isothiocyanate (TRITC) and SulfoRhodamine 101 chlorosulfonate derivative (trade name: Texas Red). Amongthese, a preferable combination is FITC and Texas Red. These labelingmaterials can be introduced into any desired portion of the sequence byperforming chemical synthesis of Probe 2 using oligonucleotides havingbonded thereto these labeling materials. Any of the fluorescent materialand quencher may be on the 5′ side.

The sequence and length of the loop region are not particularly limitedso far as a loop structure is formed when Probe 1 and Probe 2 areannealed and signals from the labeling material differ between the casewhere the loop structure is formed and the case where it is eliminated.Note that the loop region is preferably designed such that the loopregion forms neither a partial double strand with the first region andsecond region of Probe 2 nor a partial double strain within the loopregion. The length of the loop region is preferably 10 bases or more andmore preferably 20 bases or more. It is desirable that the labelingmaterial is bonded to a portion usually within 3 bases form the bothterminal bases in the loop region, preferably to the terminal bases.

The aforementioned Probe 1, Probe 2, and a sample are mixed in theconditions under which Probe 1 and Probe 2 are annealed and Probe 1 andthe target nucleic acid are annealed. However, from the aforementionedstructures of the probes, Probe 1 is annealed with the target nucleicacid preferentially than Probe 2. The order of mixing in notparticularly questioned; for example, Probe 1 and Probe 2 are mixed andthen the sample is added. Further, a premixture of Probe 1 and Probe 2may be prepared in advance. Furthermore, it is preferable that areaction mixture that does not contain Probe 1 and/or a sample is usedas a control.

Probe 1 and Probe 2 are mixed in a molar ratio of preferably 1:1. Thefinal concentration of each of Probe 1 and Probe 2 in the reactionmixture is preferably 0.1 μM or more.

After Probe 1, Probe 2, and the sample are mixed, either the mixture asit is may be exposed to the conditions in which Probe 1 and Probe 2 areannealed and Probe 1 and the target nucleic acid are annealed or themixture may be subjected to heat denaturation treatment and then exposedto the aforementioned conditions.

After leaving the aforementioned reaction mixture to stand a given time,preferably after it has reached an equilibrium state, the signal of thelabeling material is detected. More preferably, the detection of thesignal is performed with time. The aforementioned conditions include,for example, a temperature that is lower by 3 to 10° C. than the (Tm) sof Probe 1 and Probe 2. Optimal conditions can be readily determined byperforming detection of signals with time with varying temperature inseveral stages to select those that give the clearest difference fromthe control. In the case where FITC is used as a fluorescent materialand Texas is used Red as a quencher, the detection of signals isperformed by measuring intensity of fluorescence at a fluorescentwavelength of 515 nm attributable to FITC. The measurement of theintensity of fluorescence is performed using a commercially availableapparatus. The results of the measurements are shown in FIGS. 2 and 3.Those results will be described in detail in the example below.

By quantitatively detecting the signals of the labeling material, theamount of the target nucleic acid can be quantified.

The kit of the present invention is a kit that is used in order todetect the aforementioned target nucleic acid and includes Probe 1 andProbe 2. Each probe may be either a solution or a freeze-driedpreparation. Further, each probe may be either charged in a separatecontainer or in the same container as a mixture. The kit of the presentinvention may further contain a buffer for dissolving or diluting eachprobe or a sample.

The method and kit of the present invention can be advantageously usedin quantifying the amplified product in the nucleic acid amplificationreaction mixture in a real time. The kit of the present invention maycontain an oligonucleotide primer for amplifying such a target nucleicacid by a nucleic acid amplification method. The primer is charged in aseparate container from that in which each probe is contained.

EXAMPLE

Hereinafter, the present invention will be described in more detail byexamples.

Probe 1 (SEQ ID NO: 1), Probe 2 (SEQ ID NO: 2), and a targetoligonucleotide (SEQ ID NO: 3) were synthesized. The synthesis of eacholigonucleotide was requested to Japan Bio Service Co., Ltd. Thenucleotide (T) of the base 21 of Probe 2 was labeled with FITC and thenucleotide (T) of base 52 was labeled with Texas Red. Note that thetarget oligonucleotide is a partial sequence of human amylin gene.

The bases 1 to 28 of Probe 1 are complementary to the bases 6 to 33 ofthe target oligonucleotide. The bases 11 to 33 and bases 35 to 55 ofProbe 1 are complementary to the bases 52 to 74 and bases 1 to 21 ofProbe 2, respectively. Note that the bases 57 to 74 of SEQ ID NO: 2 ishomologous to the bases 6 to 23 of SEQ ID NO: 3.

Each oligonucleotide was dissolved in TE buffer to 5 μM. In a 1.5-mltube were added 2.2 μl of 10×Ex Taq buffer (Takara Shuzo Co., Ltd., Lot.A6501-1), 19.8 μl of sterilized distilled water, and 1 μl of a Probe 2solution (5 μM), which were mixed well and 23 μl of the mixture wasdispensed to each 25-μl tube for a reaction machine (Cepheid Co., SmartCycler). In each tube, 1 μl of the Probe 1 solution (5 μM) or TE bufferwas added and further 1 μl of the target oligonucleotide solution (5 μM)or TE buffer to obtain a reaction mixture. This operation was performedat room temperature.

The aforementioned reaction mixture or the aforementioned reactionmixture subjected to heat treatment at 94° C. for 3 minutes was set inthe Smart Cycler, which was adjusted to 47° C. and fluorescence at awavelength of 505 to 537 nm was measured. The results on the reactionmixture that was not subjected to the heat treatment are shown in FIG. 1and the results on the reaction mixture subjected to the heat treatmentare shown in FIG. 2.

With Probe 2 only, fluorescence was observed but in the case where Probe1 was added, the fluorescence was quenched considerably. In the casewhere the target oligonucleotide in addition to Probe 1 was added toProbe 2, fluorescence with an intermediate intensity between both thecases was observed. The results were the same regardless of presence orabsence of the heat treatment.

As described above, higher intensity of fluorescence observed in thecase where the target sequence was present than the case where no targetsequence was present confirmed that Probe 1 preferentially bonds to thetarget sequence than Probe 2 and a portion of Probe 2 was free.

INDUSTRIAL APPLICABILITY

By the present invention, a target nucleic acid can be quantified in areal time and in a simple manner. The method of the present inventiondoes not require a polymerase having a 5′→3′ exonuclease activity, sothat it can be applied to various reaction systems.

1. A method for detecting a target nucleic acid having a target sequencein a sample, comprising the steps of: (a) obtaining a first probecomprising a nucleic acid which has a specific region having a sequencecomplementary to the target sequence and a nonspecific region having asequence that is not complementary to the target sequence of the targetnucleic acid; and a second probe comprising a nucleic acid which has afirst region that is complementary to at least a portion of thenonspecific region of the first probe, a loop region that does not havea sequence complementary to the first probe, and a second region that iscomplementary to at least a portion of the specific region of the firstprobe, wherein the nucleic acid of the second probe is labeled with alabeling material generating a signal by which formation of a loop canbe detected; (b) mixing the first probe and the second probe, therebyforming a mixture wherein the loop in the loop region of the secondprobe is formed when the second probe is annealed with first probe,thereby quenching the signal from the labeling material in the absenceof a target nucleic acid; (c) adding the sample to the mixture of step(b), whereby the target nucleic acid in the sample anneals with thefirst probe, thereby releasing the second probe; and (d) detecting anincrease in the signal of the labeling material in the presence of thetarget nucleic acid compared to the signal obtained in step (b), therebydetecting the target nucleic acid, wherein the signal is quenched whenthe first probe and the second probe are annealed and not quenched whenthe first probe and the second probe are not annealed.
 2. The methodaccording to claim 1, wherein the second region of the second probe isshorter than the specific region of the first probe.
 3. The methodaccording to claim 1, wherein the labeling material comprises afluorescent material and a quencher that quenches a fluorescence of thefluorescent material when the quencher is near the fluorescent material,arranged so as to sandwich the loop region, with the fluorescence of thefluorescent material being quenched by the quencher when the first probeand the second probe are annealed to form the loop and not quenched whenthe first probe and the second probe are not annealed.
 4. The methodaccording to claim 1, wherein the detection of the increase in thesignal is performed quantitatively, thereby quantifying the targetnucleic acid.